Freezing has long been used or contemplated to preserve living cells, such as sperm, ova and blood cells; frozen storage permits the use of such cells after they have been removed or separated from the original organism. Indeed, Mantegazza, in 1866, appears to have first proposed the use of frozen banks to preserve human sperm. (Mantagazza, D., Sullo sperma umano, Rendic. Reale., Inst. Lomb. 3:183 (1866)).
For example, fresh sperm taken from a male is viable for a relatively short period of time, e.g. between about one to eight days; it is often necessary and/or advantageous to utilize the sperm long after it has been collected, e.g. for several months. Similarly, female ova taken from the womb are viable for only a short period of time. Various methods, principally freezing, have been employed to preserve the sperm and/or ova for relatively long periods of time; the need to effectively store sperm and/or ova has grown along with the increase in artificial insemination. Freezing sperm or ova permits, for example, a domestic animal breeder (for cattle, horses, swine, goats, poultry and the like) to maintain stocks of valuable sperm and/or ova for use when necessary also enables the transport of said stocks for breeding purposes which is less expensive and troublesome than transporting livestock, and permits genetically superior males to inseminate larger number of females. Artificial insemination has also been used in the human context for various medical and health reasons.
The storage and preservation of living cells such as sperm, ova and red blood cells has, however, proved to be quite troublesome because the survivability of viable cells using prior art freezing methods is often quite low. Freezing conditions are relatively harsh and thermal shock or other phenomena such as crystal formation which results from both freezing and thawing conditions often kills the cells. Therefore, maximizing the viability of thawed cells has been the goal of many researchers. For example, in the context of sperm aliquots, a thawed preparation must exhibit certain minimum fertility, typically measured by the motility of the sperm cells, or it is unlikely that the aliquot could successfully be used for artificial insemination.
The prior art discloses various methods for improving the survivability of frozen cells such as sperm and ova. U.S. Pat. No. 4,007,087 (Ericsson) discloses a sperm fractionation and storage method which claims to increase the percentage of motile sperm which survive frozen storage. Ericsson discloses a method whereby motile sperm are separated from non-motile, defective or dead sperm; the fraction containing the motile sperm is then frozen. Ericsson reports that his method increases the fertility of a sperm sample by enhancing evironmental (the ratio of total sperm to motile sperm) and viability (progressiveness of motility of the motile sperm) factors affecting the fertility of a sample, but his method does not improve the population (motile sperm count) factor which is possibly the most critical.
U.S. Pat. No. 3,791,384 (Richter et al.) discloses a method for deep freezing and thawing boar sperm which includes inactivating the fresh sperm by means of an inactivating solution which includes dextrose, dihydrate of ethlenedinitrotetra-acetic acid, sodium citrate and sodium hydrogencarbonate. Richter reports that inactivation of the sperm gives them greater power of resistance to freezing.
U.S. Pat. Nos. 4,429,542 (Sakao et al.), 4,487,033 (Sakao et al.), 3,893,308 (Barkay et al.) and 4,480,682 (Kameta et al.) all disclose different freezing methods which claim to improve the fertility of sperm and ova samples. In all of these methods, the temperature of sperm and ova samples in solution is lowered by various means which attempt to reduce the thermal shock and increase the survivability of the viable sperm and ova present. Most of these methods are, however, complex, cumbersome and expensive to utilize. Other freezing methods are also used including the "Sherman" method of rapid freezing in liquid nitrogen vapors (Sherman, J. K., Improved Methods of Preservation of Human Spermatozoa by Freezing and Freeze Drying, Fertil. Steril., 14:49-64 (1963), and the "Behrman-Sanada" method of gradual freezing (Behrman et al. Meterologous and Humologus Inseminations with Human Semen Frozen and Stored in a Liquid Nitrogen Refrigerator., Fertil. Steril. 17:457-466 (1966)).
In most freezing processes, the living cells -- prior to freezing -- are diluted with a physiologically acceptable cryoprotectant solution. In 1949, it was discovered that glycerol could be successfully used as cryoprotectant agent for the freezing of bovine sperm (Polge et al. Revival of Spermatozoa After Vitrification and Dehydration at Low Temperatures, Nature 164:666 (1949)) and since that time, the standard cryoprotectant solutions has contained glycerol, egg yolk, a sodium citrate, a buffer system, glucose, glycerine and sometimes an antibiotic or antibiotics, for example the "Ackerman" solution: Behrman and Ackerman, Am. J. Obstet. and Gynecol., 103:654-655 (1969). The effectiveness of the cryoprotectant diluent is critical to the survivability of the cells which, in the context of sperm or ova aliquots, relates directly to the fertility of the aliquot.
It has now been discovered that a cryoprotectant solution for viable cells which comprises an aqueous suspending vehicle which is physiologically acceptable to the cells and contains betaine significantly increases the survivability of the cells. For example, sperm samples frozen with the cryoprotectant solution of the present invention exhibit a dramatic increase in motility compared to samples frozen with typical cryoprotectants which do not contain betaine. The instant invention provides an effective, yet relatively simple and inexpensive cryoprotectant solution and method for freezing and preserving living cells.